The mechanisms by which synaptic vesicles in cholinergic neurons take up, bind and release acetylcholine (ACh) and the chemical entities involved in these processes remain unknown. Recently, we developed a simple, rapidly-executed technique by which changes in ACh "bound" to isolated synaptic vesicles could be monitored. By the use of this method we have discovered that isolated vesicles may be induced to "bind" or give up ACh by appropriate temperature manipulation. To this time the research has been performed mostly with synaptic vesicles isolated from guinea pig cortex. Thus, the vesicle suspensions represent mixtures of vesicles derived from cholinergic and non- cholinergic neurons. In order to determine the nature of ACh processing by vesicles derived only from cholinergic neurons a two-pronged attack has been formulated. First, we propose to study ACh processing in vesicles known to be associated with cholinergic neurons by the use of vesicles derived from the electric ray. Second, we propose to develop and refine counting procedures for isolated synaptic vesicles. These procedures in combination with determinations of vesicle-bound acetylcholine should provide a more reliable estimate of the amount of transmitter per vesicle than is now available. Estimates will also be made of the amounts of transmitter required to produce miniature end- plate potentials in the ray electric organ. The "vesicle hypothesis" will thus be tested by determining whether sufficient transmitter exists in one vesicle to produce a potential of the amplitude of a miniature end plate potential. The site of ACh "binding", whether on the vesicle membrane or within, will be studied by the use of sonicated vesicles. Finally, the in vivo release situation will be studied by the use of isolated presynaptic membranes (synaptosomal ghosts) layered onto artificial membranes brought into contact with isolated ACH-containing synaptic vesicle.